Genetic and biochemical properties of extrachromosomal genetic elements (plasmids) will be examined in bacteria with particular emphasis on colicinogenic plasmid El (ColE1), a low molecular weight derivative of the Flac plasmid (mini-F) and the antibiotic resistance plasmid R6K and RK2. The replication properties of these plasmids will be studied in Escherichia coli in vivo and in vitro with particular emphasis on the origin and directionality of replication, the essential plasmid genes for the initiation, elongation and termination of replication, incompatibility properties, and the role of the host membrane and relaxation complexes in the replication of these plasmids. The replication properties of the broad host range plasmid RK2 will be studied also in gram-negative bacteria distantly related to E. coli. Replication mutants of plasmids colE1 and R6K and conjugal transfer defective mutants of ColE1 will be examined and characterized. In vitro recombinant DNA techniques will be employed to construct joint replicons of these various plasmids and to isolate regions of plasmids containing the origin or the terminus of replication for the purpose of carrying base sequence analysis of these regions. Replication mutants of Co1E1 will be used as the basis to construct ColE1 cloning vehicles that are potentially "safer" for recombinant DNA research. Various regulatory genes of bacteriophage lambda will be attached to plasmid ColE1 with the aid of restriction endonucleases for the purpose of developing a ColE1 hybrid cloning vehicle that is more effective for gene cloning. The in vitro techniques will be carried out in strict accordance with the National Institutes of Health guidelines on recombinant DNA research.